Effect of Curcumin on Apoptosis of Acute T-Lymphoblastic Leukemia Cells induced by UMI-77 and Its Related Mechanism / 中国实验血液学杂志

AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77.@*METHODS@#T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins.@*RESULTS@#After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05).@*CONCLUSION@#Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.

Synergistic Anti-cancer Effect and Mechanism of FZKA Decoction Combined with Gefitinib on A549 Cells / 中国实验方剂学杂志

Objective::To observe the effect of Fuzheng Kangai (FZKA) decoction combined with gefitinib on the cells proliferation, apoptosis, invasion and metastasis of human lung adenocarcinoma A549 cells in vitro and in vivo, and relevant mechanisms. Method::The A549 cell proliferation of the control group, FZKA decoction groups (0.2, 0.4, 0.8, 1.6, 3.2 g·L-1), Gefitinib groups (10, 20, 40, 60, 80, 100 μmol·L-1) for 24, 48, 72 hours, and FZKA decoction (2 g·L-1) combined with Gefitinib (10 μmol·L-1) groups for 24 hours was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The changes of cell apoptosis, invasion and metastasis abilities of A549 cells were analyzed by flow cytometry, Wound Healing, transwell invasion assay. Western blot assay was used to examine the protein expressions of cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2 associated X (Bax), F-box and WD repeat domain-containing (FBW7) and myeloid cell leukemia-1 (MCL-1) in vitro. Result::Compared with control group, FZKA decoction group and Gefitinib group inhibited the cell proliferation, cell apoptosis, cell invasion and metastasis abilities in a dose-dependent and time-dependent manner, and improve the protein expressions of Bax, Caspase-3, FBW7, but decreased the protein expressions of Bcl-2, MCL-1 (P<0.05). Compared with treatment with Gefitinib alone, FZKA combined with Gefitinib inhibited the proliferation of A549 cells, and induced apoptosis more significantly (P<0.05). Compared with treatment with Gefitinib alone, the cell scratch healing and invasion abilities were significantly reduced after combined treatment (P<0.05). FZKA decoction combined with Gefitinib up-regulated Bax, Caspase-3 and FBW7 protein expressions, and down-regulated Bcl-2 and MCL-1 protein expressions compared with treatment with Gifitinib alone (P<0.05). Conclusion::FZKA decoction combined with Gefitinib can inhibit the proliferation, invasion and metastasis, and induce apoptosis on A549 cells. The mechanism may be associated with the FBW7/MCL-1 pathway.

Abt-737 enhances apoptosis of gastric cancer cells induced by MCL-1 small-molecule inhibitor UMI-77 / 中国药理学通报

Aim: To investigate the promoting effect of Bcl-2/Bcl-xL inhibitor ABT-737 on apoptosis of gastric cancer cells induced by small molecule Mcl-1 inhibitor UMI-77, and to explore its possible mechanism. Methods: The response of gastric cancer MGC-803 and HGC-27 cells to different concentrations of UMI-77 was detected by MTS assay. In the UMI-77-resistant cell lines, the effect of treatment with UMI-77/ABT-737 alone or in combination on cell viability was detected by MTS assay. The apoptotic rate and the changes of the mitochondrial membrane potential were analyzed by flow cytometry. The cleavage of caspase-9, caspase-3 and PARP-1, as well as the expression level of Bcl-2 family members and IAP proteins, were determined by Western blot. Results: Compared with MGC-803 cells, HGC-27 cells were resistant to UMI-77. Treatment with ABT-737 alone in HGC-27 cells also induced minimal level of cell death. While treatment with both agents induced much greater decreased cell viability. All the dead cells were positive for Annexin V and mitochondrial membrane potential collapsed. Caspase-9, caspase-3 and its substrate PARP-1 were cleaved. All of these proved that the sensitization effect was achieved by activating the mitochondrial apoptotic pathway. Protein levels of XIAP, cIAP1 and cIAP2 decreased after treatment with UMI-77 plus ABT-737. It also resulted in the increase of NOXA and Bcl-2 along with the decline of PUMA and Mcl-1. Conclusions: The combination of UMI-77 and ABT-737 could significantly increase the sensitivity of gastric cancer cells to the Mcl-1 small molecule inhibitor UMI-77.

Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing expression of MCL-1 / 中国病理生理杂志

AIM:To investigate the synergistic effect of toosendanin on regulating the cytotoxicity of γδ T cells to colorectal cancer cells. METHODS:γδ T cells amplified in vitro were identified by flow cytometry. Lactate dehydro-genase (LDH) release was detected to evaluate the cytotoxicity of γδ T cells and toosendanin to SW480 cells. The role of toosendanin in regulating the protein expression of Bcl-xL,Bcl-2 and MCL-1 was determined by Western blot. The effect of toosendanin on regulating the secretion of TNF-related apoptosis-inducing ligand(TRAIL) and Fas ligand(FasL) by γδ T cells was evaluated by ELISA. The mitochondrial membrane potential and apoptosis in SW480 cells treated with γδ T cells and toosendanin were analyzed by flow cytometry. The activation of caspase-9 and caspase-3 were determined by Western blot. RESULTS:CD3 and γδ T-cell receptor(TCR) were highly expressed in the γδ T cells amplified in vitro. Combina-tion with toosendanin significantly enhanced the cytotoxicity of γδ T cells to SW480 cells. Toosendanin did not influence the secretion of TRAIL and FasL secreted by γδ T cells. Toosendanin did not regulate the expression of Bcl-xL and Bcl-2 but suppressed the expression of MCL-1 in SW480 cells. In addition, enforced expression of MCL-1 obviously suppressed the synergistic effect of toosendanin on γδ T cell-induced cell death in SW480 cells. Meanwhile,co-treatment with toosendanin was able to enhance the γδ T cell-induced apoptosis and decrease of mitochondrial membrane potential. γδ T cell-depend-ent activation of caspase-9 and caspase-3 was significantly enhanced by toosendanin co-treatment in SW480 cells. CON-CLUSION:Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing the expression of MCL-1.

Effect of lipopolysaccharide on IL-6/STAT3 signal pathway in human intrahepatic biliary epithelial cells / 安徽医科大学学报

Objective To investigate the expression of IL-6/STAT3 signal pathway induced by lipopolysaccharide (LPS) on human intrahepatic biliary epithelial cells (HiBECs) in vitro.Methods The HiBECs were cultured in vitro.The cells were stimulated by LPS at different concentrations (0,0.1,1,2,4,8 μg/ml) and different time (24,48,72 h).The expression of IL-6 was measured by ELISA to determine the best concentration and time of LPS.Then we chose the best concentration and time of LPS to intervene the cells in the further experiments.The levels of mRNA expression of c-Myc and Mcl-1 were detected by fluorescence quantitative reverse transcrption PCR (RT-qRCR).The proportion of p-STAT3/STAT3 protein and the expression of c-Myc and Mcl-1 protein in HiBECs were analyzed by Western blot.Results LPS upregulated the expression of IL-6, and reached its peak at 4 μg/ml and 24 h(F=16.492,17.763,P<0.001,P<0.01).LPS increased the mRNA expression of c-Myc and Mcl-1(P<0.01).Meanwhile, The ratio of p-STAT3/STAT3 (P<0.05) protein and the expression of c-Myc (P<0.001) and Mcl-1 (P<0.05) protein were upregulated after stimulating with LPS.Conclusion The IL-6/STAT3 signal pathway can be activated by LPS in HiBECs, and LPS may promote the expression of c-Myc and Mcl-1 through this signal pathway.

Study on molecular mechanism of double hydrogen artemisinin inducing apoptosis of bile duct cancer cells / 国际检验医学杂志

Objective To study and discuss the effect of double hydrogen artesunate on Mcl-1 expression and its inducing effect on cancer cell apoptosis in the patients with cholangiocarcinoma.Methods Bile duct cancer cell lines QBC939 preserved in our hospital from June 2010 to December 2014 were randomly selected and divided into the control group and observation group for conducting experiments.The cells were cultured by using the conventional cultivation and double hydrogen artemisinin culture.Then the Mcl-1 expression and apoptosis of cancer cells were performed the statistical analysis and comparison.Results Statistical comparison showed that the expressions of MCL1-001 and-MCL1 201 at 12,24,48 h in the observation group were significantly higher than those in the control group,the comparison between groups were statistically significant (P0.05),but which at 48 h in the observation was significantly higher than that in the control group,the difference was statistically significant(P<0.05).And the mortality rate at 6,12,24,48,72 h in the observation group was significantly higher than that in the control group,the difference was statistically significant (P<0.05).Conclusion Double hydrogen artemisinin has obvious up-regulation effect on Mcl-1,moreover can effectively induces bile duct cancer cell apoptosis.

Research progress on MCL-1 and its inhibitors in hematological malignancies / 中国肿瘤临床

Myeloid cell leukemia-1 (MCL-1) protein is one of the key antiapoptotic protein members of the B-cell lymphoma-2 protein family. Overexpression of MCL-1 is closely related to not only tumor progression but also resistance to targeted therapy and traditional chemotherapeutic drug. MCL-1 and its inhibitors have been studied in recent years. The mimetics of MCL-1 endogenous ligand BH3 have resulted in significant breakthroughs. In this study, the research progress on MCL-1 and its inhibitors in hematological malignan-cies is reviewed.

Effect of Mcl-1 siRNA on TRAIL-induced apoptosis in gastric cancer cells / 安徽医科大学学报

Objective To explore the effect of Mcl-1 small interference RNA ( siRNA) on tumor necrosis factor-re-lated apoptosis-inducing ligand ( TRAIL)-induced apoptosis in gastric cancer HGC-27 cells. Methods The apop-totic rates of cells treated with TRAIL and pan-caspase inhibitor ( z-VAD-fmk) alone or combination were measured by propidium iodide (PI) method using flow cytometry. The activation of caspase-3, cleavage of PARP-1, as well as the protein level of anti-apoptotic Bcl-2 proteins Bcl-2, Bcl-XL and Mcl-1 before and after TRAIL treatment were monitored by Western blot analysis. Transfection of Mcl-1 siRNA was performed using Lipofectamine 2000 reagent. The efficiency of gene silencing was quantified by Western blot and the effect of Mcl-1 siRNA on TRAIL-induced apoptosis was measured using PI method. Results HGC-27 cells were resistant to TRAIL-induced apoptosis, and z-VAD-fmk pretreatment could block apoptosis nearly completely. Activation of caspase-3 and cleavage of PARP-1 occurred in the late stage of apoptosis. The expression levels of Bcl-2, Bcl-XL and Mcl-1 were not altered after exposure to TRAIL. Transfection with Mcl-1 siRNA could obviously downregulate the expression evel of Mcl-1 in HGC-27 cells and enhanced the sensitivity of cells to TRAIL-induced apoptosis. Conclusion Overexpression of Mcl-1 may account for the resistance of HGC-27 cells to TRAIL.ownregulationof Mcl-1 by siRNA can effectively enhance the sensitivity of HGC-27 cells to TRAIL-induced apoptosis.

Mcl-1 as a potential therapeutic target for human hepatocelluar carcinoma / 华中科技大学学报（医学）（英德文版）

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.

Mechanism of apoptosis induced by Mcl-1 inhibitor UMI-77 on gastric cancer MGC-803 cells / 安徽医科大学学报

Objective To investigate the mechanism of apoptosis induced by UMI-77 , a novel selective inhibitor of Mcl-1, in gastric cancer MGC-803 cells. Methods MGC-803 cells were treated with UMI-77 in different concen-trations for 24 h, apoptotic rates were determined by Annexin V/PI method using flow cytometry. Mitochondrial membrane potential was determined by JC-1 staining on a flow cytometer. The activation of Caspase-9, Caspase-3 and cleavage of PARP were measured by Western blot analysis. The protein level of Bcl-2, Bcl-XL and Mcl-1 was monitored by Western blot as well. Chemically synthesized Mcl-1 siRNA was transfected into MGC-803 cells using Lipofectamine 2000 Reagent. The efficacy of gene silencing was confirmed by Western blot analysis, and opoptotic rates before and after transfection was measured by flow cytometry using Annexin V/PI staining. Results UMI-77 was effective in induction of apoptosis in gastric cancer MGC-803 cells, apoptotic rates were increased in a dose-de-pendent manner. Mitochondrial membrane potential was collapsed after UMI-77 treatment. Activation of Caspase-9, Caspase-3 and cleavage of PARP occurred at 24 h (P<0. 05). The expression level of Bcl-2 and Bcl-XL were not altered after exposure to UMI-77 , while Mcl-1 was down-regulated after 12 h ( P<0. 05 ) . Transfection with Mcl-1 siRNA successfully decreased the expression level of Mcl-1 in MGC-803 cells ( P<0. 05 ) and blocked apoptosis induced by UMI-77 ( P<0. 05 ) . Conclusion UMI-77 induces apoptosis through activation of the intrinsic path-way in gastric cancer MGC-803 cells, and knocking down Mcl-1 expression abrogates apoptosis by UMI-77.

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis / 中国病理生理杂志

AIM:To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference .METH-ODS:The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang , H37Rv, H37Ra and BCG.Mcl-1-shRNA was applied to the mouse model of infection , and the control groups were set up .On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected .The expression of Mcl-1 at mRNA and protein levels was deter-mined by real-time PCR and Western blot .The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry . RESULTS:The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05).The expres-sion of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group ( P<0.05 ) . Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice .CONCLUSION: The Mcl-1 ex-pression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tu-berculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis .

Effect of Mcl-1 signaling pathway blockers on apoptosis of mouse macro-phages infected with Mycobacterium tuberculosis H37Rv / 中国病理生理杂志

[ ABSTRACT] AIM: To explore the effects of Mcl-1 signal pathway blockers on Mcl-1 expression, macrophage apoptosis and Mycobacterium tuberculosis in the model of mice infected with Mycobacterium tuberculosis H37Rv.METH-ODS:A mouse infection model was established by intraperitoneal injection of H37Rv suspension.The signaling pathway blockers AG490, PD98059 and LY294002 for JAK/STAT, MAPK and PI3K, respectively, were intraperitoneally injected into the mice infected with H37Rv.Cell acid-fast staining was used to observe whether the mouse peritoneal macrophages infected with H37Rv were successfully established.Immunocytochemical method was employed to detect Mcl-1 expression in the mouse peritoneal macrophages infected with H37Rv.The apoptotic rate in each group was measured by flow cytomer-ty.The scavenging capacity of apoptotic macrophages against H37Rv was determined by Mycobacterium tuberculosis colony counting.RESULTS:The result of cell acid-fast staining revealed the existence of dispersive arrangement of red short anti-acid Mycobacterium tuberculosis within infected macrophages.The result of cell immunocytochemistry showed strongly posi-tive expression of Mcl-1 protein in H37Rv infection group, AG490 treatment group and LY294002 treatment group, weakly positive expression of Mcl-1 protein in PD98059 treatment group, and negative expression of Mcl-1 protein in control group. The result of flow cytometry found that the macrophage apoptotic rate in H37Rv infection group was higher than that in con-trol group, while that in PD98059 treatment group was high than that in other groups with statistically significant differences (P<0.05).The result of Mycobacterium tuberculosis colony counting showed that PD98059 treatment had the most signifi-cant inhibitory effect on H37Rv strain.CONCLUSION: Mcl-1 signaling pathway blockers increase the apoptotic rate of macrophages infected with Mycobacterium tuberculosis H37Rv and inhibit the growth of Mycobacterium tuberculosis by inhibi-ting the signaling pathways of JAK/STAT, MAPK and PI3K, among which the MAPK has the most obvious interfering effect on Mcl-1, and leads to the highest apoptotic rate of infected macrophages and the strongest bacteriostasis.

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer / 中国病理生理杂志

AIM:To investigate the effect of microRNA ( miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS:miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls.MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells.Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b.Mcl-1 expression vector was constructed , and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay .RESULTS:Down-regulation of miR-193b was found in breast cancer patients .The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells.The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection.The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was sig-nificantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer .

Antitumor effect of imperatorin enhances cytotoxicity of doxorubicin to HeLa cells / 中国病理生理杂志

AIM:To determine whether imperatorin would enhance the effect of doxorubicin therapy on cervi -cal cancer in vitro.METHODS:The viability of HeLa cells treated with imperatorin and doxorubicin was determined by MTT assay in vitro.The expression of Bcl-2 protein family ( Mcl-1, Bcl-2, Bcl-xL, Bad and Bax ) in HeLa cells treated with imperatorin and doxorubicin was evaluated by Western blot analysis .The apoptosis and mitochondrial membrane poten-tial (ΔΨm ) in the HeLa cells treated with imperatorin and doxorubicin were analyzed by flow cytometry .A Mcl-1 expres-sion vector was constructed , and its role in the cytotoxicity of imperatorin plus doxorubicin to HeLa cells was detected by MTT assay.RESULTS: Addition of imperatorin significantly enhanced the cytotoxicity of doxorubicin to HeLa cells in vitro.Mcl-1 expression was down-regulated by imperatorin but was not influenced by doxorubicin in the HeLa cells .A com-bination of imperatorin and doxorubicin induced apoptosis and ΔΨm collapse more significantly compared with the treatment with imperatorin or doxorubicin alone .Furthermore, the imperatorin-induced sensitization for doxorubicin cytotoxicity to HeLa cells was abolished by the transfection with Mcl-1 expression plasmid .CONCLUSION:The combination of doxoru-bicin with imperatorin enhances the antitumor effect of doxorubicin on cervical cancer cells via targeting Mcl -1.

Specificity of screening of short hairpin RNA targeting Mcl-1 gene in Raw264.7 cells / 中国免疫学杂志

Objective:To transfect Mcl-1shRNA into Raw264.7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw 264.7.Methods: Specific shRNA was transfected into murine macrophage cell line Raw 264.7 via lipofectamine.Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection ,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed.Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level ,with higher silencing effects than those of the normal group ,the lipofectamine group and the negative control group.There were statistically significant differences among them ( P<0.05 ).Among the three pairs of specific shRNA fragments corre-sponding to different sites ,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins.Conclusion:RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw 264.7 and greatly downregulate the expression of Mcl-1protein.Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.

miR-363 downregulates Mcl-1 expression and induces apoptosis in HepG2 cells / 中国病理生理杂志

AIM:To determine the effect of microRNA-363(miR-363) on HepG2 cells.METHODS:Bioin-formatic analysis was conducted to identify if the Mcl-1 was regulated by miR-363.The expression of Mcl-1 and miR-363 was detected by real-time PCR in normal liver cell line LO2 and hepatocellular carcinoma cell line HepG2, Huh7 and PLC. miR-363 was transfected into the HepG2 cells, and then the level of Mcl-1 was measured.The relative viability was evalua-ted by MTT assay after the HepG2 cells were transfected with miR-363, and the apoptosis was analyzed by flow cytometry with Annexin V/PI staining.RESULTS:Bioinformatic analysis identified that there was a putative target site in the Mcl-1 mRNA for miR-363.Transfection of miR-363 mimics suppressed Mcl-1 expression in the HepG2 cells.Transfection of miR-363 mimics inhibited the cell viability as well as inducing cell apoptosis in HepG2 cells.CONCLUSION: Over-ex-pression of miR-363 significantly inhibits the cell viability and induces apoptosis in HepG2 cells, and the mechanism may be related to the downregulation of Mcl-1 caused by miR-363 transfection.

The effects of siRNA targeting Mcl-1 on biological behavior of salivary adenoid cystic carcinoma SACC-2 cells / 实用口腔医学杂志

Objective:To explore the effect of siRNA targeting myeloid cell leukemia-1(Mcl-1)on the biological behavior of salivary adenoid cystic carcinoma cells.Methods:The chemically synthesized Mcl-1-siRNA was transfected into salivary adenoid cystic carci-noma SACC-2 cells.The expression levels of Mcl-1-mRNA and Mcl-1protein were examined by Real-time PCR and western blotting respectively.MTT assay,transwell chamber and flow cytometry were used to determine the effect of Mcl-1-siRNA on SACC-2 cell pro-liferation,migration and apoptosis.Results:Compared with the control group,liposome group and NC-siRNA group,SACC-2 cell proliferation rate of Mcl-1-siRNA group was obviously slowed down.48 h after transfection,the migration of SACC-2 cells in Mcl-1-siRNA group(39 ±9.0)were lower than that in control group(69 ±6.0).The apoptosis rate of Mcl-1-siRNA group(8.6%)was sig-nificantly higher than that in control group(1.9%).Conclusion:Silence Mcl-1 can inhibit cell proliferation and migration and pro-mote apoptosis of salivary adenoid cystic carcinoma cells.

Experiment study on screening efffective Mcl-1-targeted siRNA squence in gastric cancer cells / 实用医学杂志

Objective To detect the expression of Mcl-1 gene in gastric cancer cell lines SGC7901 and MGC-803, and to screen the most effective Mcl-1-targeted siRNA sequence. Methods Mcl-1 expression was evaluated in human gastric cancer cell lines SGC7901 and MGC-803 by RT-PCR. Four segments of siRNAs (siRNA1, siRNA2, siRNA3 and siRNA4) targeting Mcl-1 mRNA and a no-sense control segment were designed by bioinformatic technology . Mcl-1 specific siRNAs were transfected transiently into SGC7901 and MGC-803 cells by using lipofectamine 2000 . After transfected 24 , 48 and 72 h , quantitative real-time PCR was applied to detect the mRNA expression of Mcl-1 and western-blot analysis was applied to detect the protein expression. Results Mcl-1 gene was expressed in both SGC7901 and MGC-803 cells. Overall, siRNA1 exhibited the best inhibitory effect after being transfected for 48h. The inhibition rate of mRNA level in SGC7901 group and MGC-803 group was 73.8%and 67.6%, and the inhibition rate of protein level in SGC7901 group and MGC-803 group was 79.3%and 96.1%. Conclusion Mcl-1 specific siRNA sequences were successfully designed, and siRNA1 was selected as the most effective sequence, which can simultanandeously inhibit the expression of Mcl-1 in GC7901 and MGC-803 cells.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

MicroRNA-29a regulates apoptosis induced by lipopolysaccharide in THP-1 cells / 中华急诊医学杂志

Objective To investigate the effects of microRNA-29a (miR-29a) on lipopolysaccharide (LPS)-induced apoptosis in human monocytes THP-1 cells in order to understand the molecular mechanisms.Methods Human monocytes THP-1 cell line were exposed to LPS after transfected with miR-29a inhibitors (100 nmol/L) or just transfected with miR-29a mimic (100 nmol/L) by lipofectamine RNAiMAX.Flow cytometry (FCM) was used to detect the cell apoptosis.Real-time RT-PCR was employed to measure expressive levels of the gene Bcl-2 and Mcl-1.The luciferase assay was performed in HEK293T cells,which were co-transfected with plasmid DNA and miRNA by using Lipofectamine 2000.Statistical analysis carried out by using SPSS 13.0 software for One-way ANOVA and Student' s t test.Results Transfection with miR-29a mimics for 48 h increased apoptosis rate and significantly reduced the expressions of Bcl-2 and Mcl-1 in THP-1 cells in comparsion with the control group.The apoptosis rate also raised in THP-1 cell stimulated by LPS for 24 h followed by LPS stimulation for 24 h,the apoptosis rate was decreased in comparison with the LPS group.In addition,our luciferase assay data showed that HEK293T cells cotransfected with miR-29a mimics and Bcl-2 3 ' UTR-Wt or Mcl-1 3' UTR-Wt plasmid significantly reduced the luciferase activity compared with the control group.Conclusions The miR-29a may regulate apoptosis by targeting the genes Bcl-2 and Mcl-1,and miR-29a may play a pivotal role in the process of apoptosis in immune cells.